CHANGES IN THE LACTATE AND MALATE DEHYDROGENASE ISOENZYME PATTERNS OF CHICKEN EMBRYO BRAIN AND RETINA

Abstract— Lactate dehydrogenase and malate dehydrogenase isoenzyme patterns of chicken brain and retina have been investigated by cellulose acetate electrophoresis, during embryonic and post-hatching development. The proportion of M-type lactate dehydrogenase subunits decreases significantly in brain and retina with development. A marked increase in the H-type subunits is observed in retina. Lactate dehydrogenase isoenzyme distribution appears to change in both organs in parallel with the metabolic changes of differentiation.
Malate dehydrogenase isoenzyme patterns do not reveal any consistent change of the ratio between the mitochondrial and cytoplasmic forms.     

Separation of rat lactate dehydrogenase isoenzyme C4 from other isoenzymes by affinity and ion-exchange chromatography.

Lactate dehydrogenase C, an isoenzyme composed of C polypeptide subunits and found only in mature testes and spermatozoa, differs kinetically, chemically and immunologically from the five common isoenzymes of lactate dehydrogenase, each of which is a tetramer of A and/or B subunits. In the rat lactate dehydrogenase C exists in two molecular forms, isoenzymes C4 and A1C3. In addition to these two forms of lactate dehydrogenase C, rat testicular homogenate contains all the five isoenzymes of A and B type. Purification of isoenzyme C4 requires its separation from the other six isoenzymes, of which isoenzymes A1C3 and A3B1 are the most difficult ones to separate. In the present study isoenzyme A3B1, along with other enzymes, was separated from isoenzyme C4 by AMP-Sepharose chromatography by using a gradient of increasing concentration of NAD+-pyruvate adduct. In the next step, isoenzyme A1C3 was separated from isoenzyme C4 by DEAD-cellulose chromatography, resulting in a pure lactate dehydrogenase isoenzyme C4 preparation.     

Lactate dehydrogenase isozymes in the preimplantation rabbit embryo

The rabbit zygote on day 1 of development contains predominantly A-type subunits of lactate dehydrogenase. During cleavage, the isozyme pattern shows a considerable increase in the amount of B subunits present. These findings indicate that the synthesis and metabolism of lactate dehydrogenase in the rabbit embryo are markedly different from those of the mouse embryo.This research was supported by the National Institute of Child Health and Human Development Grant 03071.     

Development of glial cells in primary cultures: Energy metabolism and lactate dehydrogenase isoenzymes

Primary cultures of glial cells prepared from brains of newborn rats were grown for periods of 1–5 weeks. After a proliferative phase of between 2 and 3 weeks, the cultures were maintained in stationary phase, during which a significant increase of oxygen consumption and of the activities of lactate dehydrogenase, succinate dehydrogenase, and mitochondrial glycerolphosphate dehydrogenase could be observed. Furthermore, qualitative changes in the lactate dehydrogenase isoenzyme pattern were found with time, characterized by a shift toward an enhanced synthesis of H subunits. A similar development was found in comparing the LDH isoenzyme pattern in the brain of 15-day-old rat embryo with those of newborn and adult rat brains. It is suggested that some aspects of maturation of glial cells in culture are comparable to those occurring in whole brain in vivo, namely a shift towards an enhanced aerobic metabolism.     

Changes in the lactate dehydrogenase isoenzyme pattern during differentiation of rabbit bone-marrow erythroid cells.

1. Differentiation and maturation of rabbit bone-marrow erythroid cells was accompanied by a 15-fold decrease in lactate dehydrogenase activity from approx. 0.1pmol of NADH utilized/min per cell in basophilic cells to 0.007 pmol of NADH/min per cell in reticulocytes. 2. In early cells, cell division takes place with a corresponding decrease in cell volume, but the concentration of lactate dehydrogenase remains almost constant. 3. When cell division ceases, qualitative as well as quantitative changes in the lactate dehydrogenase isoenzyme pattern become apparent and reticulocytes were found to contain almost exclusively the H4 isoenzyme, whereas early erythroblasts contained also the M4 and hybrid isoenzymes. 4. Extracts from a lysosome-enriched subcellular fraction of bone-marrow erythroid cells specifically degraded the M4 isoenzyme in vitro, but the H4 form was stable. It is suggested that lysosomal enzymes are involved in bringing about the observed changes in lactate dehydrogenase isoenzyme patterns in vivo.     

The excretion of lactate dehydrogenase in human urine after ingestion of aspirin

1. Cells present in normal human urine contain 5-10% of the total lactate dehydrogenase excreted. The enzyme released from these cells by ultrasonication contained a distribution of isoenzymes similar to that found in the bulk of the urine and it is suggested that these cells are the main source of urinary lactate dehydrogenase. 2. Cells were thoroughly washed before examination so it is unlikely that the enzyme found in urinary sediment was simply adsorbed. In addition, full recoveries of added lactate dehydrogenase isoenzymes LDH(1) and LDH(5) showed that adsorption did not occur. 3. Most of the cells in normal urine are of the non-squamous epithelial type and their excretion is greatly increased after the ingestion by the subject of 3g. of aspirin. The possible origin of these non-squamous cells from the kidney is discussed. 4. Starch-block electrophoresis and relative activity measurements of lactate dehydrogenase excreted after the subject had taken aspirin show that the enzymes present in urine and cells are very similar, confirming the conclusion reached above (point 1). They have slightly more M subunits than the normal, shown particularly as an increase in isoenzyme LDH(2). The isoenzyme pattern is like that of the kidney medulla and the possible reasons for this are discussed in terms of the concentration of salicylic acid in various parts of the kidney. 5. The results confirm the previous suggestion that the kidney is the main source of urinary lactate dehydrogenase.     

Substrate inhibition of soluble and bound lactate dehydrogenase (isoenzyme 5)

Substrate inhibition of chicken lactate dehydrogenase (EC 1.1.1.27) isoenzyme 5, was studied with the enzyme in the soluble phase and bound to muscle subcellular particulate structures. Inhibition studies were performed by incubating bound or soluble enzyme with NAD⁺ prior to measuring the reaction with a stopped-flow technique at 40 °C and a concentration of enzyme of 10^?7m. The value of V for soluble lactate dehydrogenase was 610 nmoles per sec, and for the bound enzyme it was 262. k_m (pyruvate) values were similar for both enzymes. Under our experimental conditions, up to 73% inhibition of the soluble enzyme was observed. On the other hand, there was no detectable inhibition of bound lactate dehydrogenase. It is suggested that the resistance to substrate inhibition of bound lactate dehydrogenase may possibly be due to the prevention of dissociation of the enzyme into monomeric or other subunits because of attachment to the particulate structures.     

Limited proteolysis of bovine muscle and heart lactate dehydrogenase is inhibited by phospholipid liposome interaction

Limited proteolysis of phospholipid complexes of heart and muscle bovine lactate dehydrogenase by trypsin and chymotrypsin has been studied under nondenaturing condition at pH 7.5. Chymotrypsin cleaves the polypeptide chain of heart and muscle lactate dehydrogenase into two principal fragments and LDH subunits were protected by lipids towards the proteinase attack. Enzymatic activity of heart and muscle lactate dehydrogenase was abolished by limited proteolytic cleavage. In complexes, both isoenzymes were protected against proteinases attack by lipids.     

Immunochemical studies on lactate dehydrogenase A subunit deficiencies.

In lactate dehydrogenase (LDH) A subunit deficiency, is there not only a lack of activity but also a lack of subunit production? We demonstrated three remarkable points to answer this question: There are no proteins that immunologically react with anti-A subunits. There are no heterotetramers that react with anti-B subunits. B subunits seem to be genetically produced at normal level, and all of them form only one isoenzyme, LDH-B4. From these points, we concluded that there is a complete lack of A subunit production or production of incomplete A subunits that can neither react with anti-A subunits nor form heterotetramers.     

Lactate and malate dehydrogenases in the muscles and male genital tract of the rabbit.

Average lactate dehydrogenase (LDH) isoenzyme patterns the content of H subunits, total LDH activity, total malate dehydrogenase (MDH) activity and the m- MDH/s-MDH ratio were determined in twelve muscles and the male genital tract of the rabbit. LDH(1) was the predominant form in the heart, soleus and masseter muscles, LDH(3) in the lingual muscles and LDH(5) in the other muscles analysed. In the muscles, an increase in the percentual proportion of M subunits was accompanied, by a proportional increase in total LDH activity and a decrease in total MDH activity, especially m-MDH. LDH isoenzyme patterns and LDH and MDH activities are useful for obtaining some idea about the proportion of individual muscle fibres. Activity accounted for by H subunits was roughly the same in all the muscles analysed, indicating that the synthesis of H subunits is independent of the type of muscle fibre and of the oxygen supply of the muscular tissue, and also that isoenzymes composed chiefly of H subunits are not localized preferentially in the mitochondria. Similar relationships between LDH isoenzymes and LDH and MDH activities were found in the testicular and epididymal tissues. The tests and the head of the epididymis mainly contain LDH isoenzymes composed of H subunits. The total LDH activity in these tissues is relatively low and their MDH activity is relatively high compared with the body and tail of the epididymis. The proportion of H subunits in the ampulla, the seminal vesicles, the coagulating glands and the prostate is also high. Cowper's glands have a high LDH(5) and LDH(4) concentration. One of two LDHx isoenzymes were found in the testes and spermatozoa.     

Isolation of human lactate dehydrogenase isoenzyme X by affinity chromatography.

Human isoenzyme LDH-X (lactate dehydrogenase isoenzyme X) was isolated from seminal fluid of frozen semen samples by affinity chromatography by using oxamate-Sepharose and AMP-Sepharose. In the presence of 1.6 mM-NAD+, isoenzyme LDH-X does not bind to AMP-Sepharose, whereas the other lactate dehydrogenase isoenzymes do. This is the crucial point in the isolation of isoenzyme LDH-X from the other isoenzymes. The purified human isoenzyme LDH-X had a specific activity of 146 units/mg of protein.     

Lactate dehydrogenase ontogeny,paternal gene activation,and tetramer assembly in embryos of brook trout,lake trout,and their hybrids

Measurement of lactate dehydrogenase in reciprocal hybrids of trout during development revealed that a maternal effect was involved in the regulation of enzyme levels until resorption of the yolk sac was completed. Malate dehydrogenase specific activities were the same in these embryos and larvae. The more negatively charged B subunits of LDH predominated during early stages of embryogenesis in lake trout and brook trout with an increase in synthesis of A subunits evident as development progressed. Activation of the paternal A gene in reciprocal hybrids occurred at a relatively late stage with the LDH subunit specific to the retina appearing after hatching. Analysis of brook trout progeny from a cross of parental types with a variant and wild-type B subunit suggested nonrandom LDH tetramer assembly which may be genetically controlled.This study was supported by National Science Foundation Grant GB-7271.     

Glycolytic enzymes in polyamine-treated bovine retina

The retina is characterized by glycolysis under aerobic conditions, mediated by lactate dehydrogenase isoenzyme-5 (LDH-5) as well as by the soluble isoenzyme of malate dehydrogenase. Bovine retina LDH and MDH isoenzymes and their activities were studied after polyamine treatment. Our results showed that LDH-5 isoenzyme presented the highest activity in untreated as well as in putrescine-treated retina. Decreased activity was present when the retina was treated with spermidine or spermine. It was demonstrated that retinic LDH-5 had a high affinity for lactate which enabled the isoenzyme to be more effective than the other LDH isoenzymes in the conversion of NADH to NAD. Therefore, the putrescine enhancing LDH-5 activity appeared to be capable of stimulating NAD-mediated rhodopsin regeneration. Putrescine induced a marked increase of both MDH isoenzymes--soluble (s-MDH) and mitochondrial (m-MDH), while spermine and spermidine mostly affected the soluble form of the enzyme. Putrescine induced a three-fold increase in s-MDH and m-MDH activities, while spermine and spermidine induced a four to five-fold increase in s-MDH. These results document the differential effects of polyamine treatment on LDH and MDH isoenzyme activities.     

Prenatal changes of lactate dehydrogenase isoenzyme patterns and activity in bovine tissues.

Isoenzyme patterns, total lactate dehydrogenase (LDH, E.C. 1.1.1.27) activity and H and M subunit activity were determined in the tissues of Czech Spotted bovine foetuses. Total LDH activity rose in the skeletal muscles throughout the whole of the prenatal period. In the viscera it usually attained the maximum at a foetal length of 66.7 cm. Differences in the isoenzyme patterns of the various organs of an 8.1-cm foetus were relatively small (41.9--66.1% H subunits). In the heart and kidneys, in which LDH1 and LDH2 markedly predominate in adulthood, the isoenzyme pattern resembled the adult one at a length of only 13.3 cm, but in the liver, spleen and lungs not until 66.7 cm. The proportion of H subunits also rose in the part of the gastrointestinal tract where secretory and resorptive activity predominate (the abomasum, the small and the large intestine). Conversely, it fell in organs concerned mainly with the mechanical processing of food (the rumen, reticulum and omasum). The proportion of M subunits rose in all the skeletal muscles up to a foetal length of 66.7 cm. Later on, differentiation into muscles in which M subunits predominated (the longissimus dorsi and the triceps brachii), into muscles with approximately the same proportion of H and M subunits (the iliopsoas) and to muscles with a preponderance of H subunits (the masseter and the muscular part of the diaphragm) occurred.     

Rice cytosolic glyceraldehyde 3-phosphate dehydrogenase contains two subunits differentially regulated by anaerobiosis

Rice cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is composed of two subunits of different molecular weights. Cytosolic GAPDH activity and protein both decreased immediately after transfer of 48-h rice seedlings to anaerobic conditions. Subsequent increase in activity and protein was accompanied by a change in isoenzyme profile and was preceded by an increase in steady-state messenger levels. One and two-dimensional electrophoretic analyses of in vivo and in vitro labeled GAPDH suggested that the change in isoenzyme profile under anaerobic conditions is due to preferential synthesis of one of the two GAPDH subunits caused by a specific increase in its mRNA.     

Factors affecting the lactate dehydrogenase isoenzyme pattern of cultured kidney-cortex cells

1. The lactate dehydrogenase isoenzyme pattern of cultured calf kidney-cortex cells was correlated to growth phase, changes in oxygen supply, mean generation time and changes in nutritional supply. 2. During culture of free cells and intact explants the lactate dehydrogenase isoenzyme pattern changed towards a dominance of isoenzymes containing the M subunit. 3. Of the shift in monomer proportion, 58% occurred during the lag phase and 42% during the initial part of the exponential growth phase. During the stationary phase the shift in monomer proportion reversed slightly. It was possible to relate the observed shift in monomer proportion to the glycolytic rate. 4. Factors that depressed glycolysis decreased the shift in monomer proportion. Oxygen was found to limit the decrease in the H subunit/M subunit ratio caused by anaerobic culture in vitro. 5. The results obtained support the view that the altered lactate dehydrogenase isoenzyme pattern of urine in renal ischaemia may be explained by anaerobic changes in the lactate dehydrogenase isoenzyme pattern of cortical tubule cells.     

A study of lactate dehydrogenase levels and turnover rates during postnatal development in the rat

Specific radioimmunoassays for lactate dehydrogenase A and B subunits have been employed to quantify cellular contents of these proteins more precisely than hitherto possible and to monitor changes during postnatal development. Liver, skeletal muscle, heart muscle and kidney cortex all demonstrated alterations in cellular levels of lactate dehydrogenase subunits over the first 56 days of life, the particular pattern being specific to each tissue. Studies on the turnover of lactate dehydrogenase in vivo and in vitro indicated that the developmental changes in total lactate dehydrogenase content in liver and kidney were regulated at some point(s) during both the biosynthesis and the degradation of the proteins.     

Isoenzyme spectrum of lactate dehydrogenase, malate dehydrogenase, esterase and acid phosphatase of rat brain cells at different times after external 1 Gy gamma irradiation]

The influence of external single gamma-irradiation with a dose of 1 Gy on the isoenzyme composition of lactate dehydrogenase, malate dehydrogenase, esterase and acid phosphatase in the cytoplasm of rat brain cells has been investigated. Irradiation was shown to cause differently directed changes in the ratio of the isoenzymes under study at different times after exposure. The isoenzyme spectrum of lactate dehydrogenase and malate dehydrogenase was shown to be normalized on day 30 after irradiation, whereas the isoform composition of esterase and acid phosphatase was not stabilized at that time.     

Adsorptive pinocytosis of 125I-labelled lactate dehydrogenase isoenzymes H4 and M4 by rat yolk sacs incubated in vitro.

The pinocytic uptake of 125I-labelled porcine lactate dehydrogenase isoenzymes H4 and M4 by 17.5-day rat visceral yolk sac incubated in vitro was saturable and binding obeyed Michaelis-Menten kinetics. The uptake characteristics of the two isoenzymes were very similar. For the H4 and the M4 isoenzymes, the dissociation constants of the protein-plasma-membrane complex were 0.62 microM and 0.84 microM respectively, and the maximum rates of uptake 0.13 and 0.26 nmol/mg of yolk-sac protein per h respectively. These findings contrast with those from studies in vivo, which show the M4 form is taken up by rat liver sinusoidal cells at a much higher rate than the H4 form, and point to different recognition systems for the adsorptive pinocytosis of simple non-conjugate proteins in yolk-sac epithelial cells and liver sinusoidal cells. Competition experiments indicate that binding of the H4 isoenzyme to the yolk-sac cells is restricted to hydrophobic interactions, whereas the binding of the M4 isoenzyme involves hydrophobic as well as positively charged sites on the protein molecules.     

Serum Enzyme Activity of Newborn Calves,Pigs, and Lambs

Serum activities of aspartate aminotransferase (AspAT = GOT), alanine aminotransferase (AlAT = GPT), and total lactate dehydrogenase (LDH) have been investigated in newborn calves, pigs, and lambs. In the two latter species the LDH isoenzyme distribution in serum was also studied. Blood samples were taken at frequent intervals from birth to 48–72 hrs. post partum. Calves and pigs were born with very low serum enzyme values, whereas lambs showed a picture more similar to what has been reported in human infants. In all species a marked temporary enzyme increase occurred during the first 24–48 hrs. This elevation was found not to be due to colostrum feeding, since a parallel increase was found in starved animals. Possible regulating mechanisms are discussed. The LDH isoenzyme pattern proved to be more stable than total LDH in the early post-natal period. The percentage isoenzyme distribution, however, showed characteristic differences from that found in adult animals of the same species.